The long-term objective of this project is to understand the molecular mechanisms involved in facilitative glucose transport. The recent discovery that HIV protease inhibitors are capable of selectively inhibiting GLUT4, the major insulin responsive glucose transporter, is the first report that it is possible to selectively and reversibly inhibit the activity of a single facilitative glucose transporter isoform. The mechanism by which this inhibition occurs remains unknown. The specific goals of this research are to investigate the mechanistic basis for the inhibition of GLUT4 by HIV protease inhibitors and determine whether the in vitro effects on GLUT4 can be replicated in vivo, leading to acute and reversible insulin resistance. First, a careful kinetic analysis of the inhibition process will be conducted using GLUT4 heterologously expressed in Xenopus oocytes. Next, the site of HIV protease inhibitor binding to GLUT4 will be determined through photolabeling of the transporter in Xenopus oocytes and/or 3T3-L1 adipocytes using a synthesized reactive retroviral protease inhibitor derivative. Modified protein will be analyzed by Surface Enhanced Laser Desorption/Ionization (SELDI) mass spectrometry. Finally, the ability of HIV protease inhibitors to acutely and reversibly cause insulin resistance in vivo will be investigated by measuring glucose disposal under euglycemic hyperinsulinemic clamp conditions in both normal and diabetes susceptible rodents. A better understanding of the mechanism by which the activity of facilitative glucose transporters can be acutely modulated in an isoform specific manner will provide a new means of studying glucose homeostasis in normal individuals and those with disorders of glucose regulation, such as diabetes mellitus. The results of this research may also facilitate the development of newer HIV protease inhibitors that maintain their efficacy in HIV treatment while avoiding their adverse metabolic consequences.